Gene expression is a complex set of events coordinated by a plethora of sequence-specific transcription factors (TFs) that bind and recognise regulatory elements of genes in a combinatorial fashion, and interface with chromatin structures. NF-Y is an ubiquitous eukaryotic TF composed by evolutionary conserved subunits (NF-YA, NF-YB and NF-YC), all necessary for specific binding of the CCAAT box, a widespread element occurring in 30% of genes promoters. NF-Y binding is critical in transcriptional regulation, establishing a permissive chromatin conformation and enabling master TF binding. Genome-wide analyses of TF locations in vivo unveiled NF-Y partnership with a defined set of TFs, some of them with precise binding-site (TFBS) arrangements with respect to the CCAAT box. Analysis of promoters of cancer “signature” genes pointed toward NF-Y and E2F TFBSs as the most enriched motifs in genes overexpressed in human tumours. Their significant co-localization in vivo further suggests NF-Y and E2Fs might functionally interact by building a landing platform for co- activators. With the long-term goal of controlling such TF interactions in cancer, in this thesis we initially focused on E2F structure, and then extended our studies to NF-Y interactions on DNA. The E2F family of TFs, originally identified with E2F1 as master regulators of cell-cycle progression, bind DNA as heterodimers with the structurally related DP subunits. Their hallmark features consist of a DNA Binding Domain (DBD) connected through a short linker to the Coiled- Coil-Marked Box (CC-MB) dimerization domain, also involved in Rb tumour suppressor proteins interactions. As structural information is only available for the separated domains, we set out to obtain a coherent view of the linked domains on DNA, and produced the ensembles of DBD-CC-MB portions of E2F1/DP1. To structurally characterise the E2F1/DP1/DNA complex we applied an integrated approach that combines in-solution (SAXS) analyses with Molecular Dynamics simulations. We provide evidence for a high level of flexibility in the overall relative topology of the heterodimer, including in a newly identified part stabilizing DNA contacts. Next, we applied EMSA experiments to characterise DNA binding of the CDC2 cell-cycle promoter, shown to be functionally dependent from both E2F and NF-Y. While results obtained indicate non-cooperative DNA association of NF-Y and E2F1, suggesting alternative mechanisms to elicit transcriptional synergy, we observed efficient assembly of stable ternary complexes. Together, our results and tools obtained in this work will be instrumental for future structural studies involving multi-protein complex assembly on DNA, and for defining the molecular bases of TFs interactions involved in architectural organisation of gene promoters.

STRUCTURAL DETERMINANTS OF NF-Y INTERACTION WITH PARTNER TRANSCRIPTION FACTORS ON DNA / D. Saad ; tutor: N. Gnesutta ; co-tutor: R. Mantovani. - : . Dipartimento di Bioscienze, 2021 Apr 22. ((33. ciclo, Anno Accademico 2020.

STRUCTURAL DETERMINANTS OF NF-Y INTERACTION WITH PARTNER TRANSCRIPTION FACTORS ON DNA

D. Saad
2021

Abstract

Gene expression is a complex set of events coordinated by a plethora of sequence-specific transcription factors (TFs) that bind and recognise regulatory elements of genes in a combinatorial fashion, and interface with chromatin structures. NF-Y is an ubiquitous eukaryotic TF composed by evolutionary conserved subunits (NF-YA, NF-YB and NF-YC), all necessary for specific binding of the CCAAT box, a widespread element occurring in 30% of genes promoters. NF-Y binding is critical in transcriptional regulation, establishing a permissive chromatin conformation and enabling master TF binding. Genome-wide analyses of TF locations in vivo unveiled NF-Y partnership with a defined set of TFs, some of them with precise binding-site (TFBS) arrangements with respect to the CCAAT box. Analysis of promoters of cancer “signature” genes pointed toward NF-Y and E2F TFBSs as the most enriched motifs in genes overexpressed in human tumours. Their significant co-localization in vivo further suggests NF-Y and E2Fs might functionally interact by building a landing platform for co- activators. With the long-term goal of controlling such TF interactions in cancer, in this thesis we initially focused on E2F structure, and then extended our studies to NF-Y interactions on DNA. The E2F family of TFs, originally identified with E2F1 as master regulators of cell-cycle progression, bind DNA as heterodimers with the structurally related DP subunits. Their hallmark features consist of a DNA Binding Domain (DBD) connected through a short linker to the Coiled- Coil-Marked Box (CC-MB) dimerization domain, also involved in Rb tumour suppressor proteins interactions. As structural information is only available for the separated domains, we set out to obtain a coherent view of the linked domains on DNA, and produced the ensembles of DBD-CC-MB portions of E2F1/DP1. To structurally characterise the E2F1/DP1/DNA complex we applied an integrated approach that combines in-solution (SAXS) analyses with Molecular Dynamics simulations. We provide evidence for a high level of flexibility in the overall relative topology of the heterodimer, including in a newly identified part stabilizing DNA contacts. Next, we applied EMSA experiments to characterise DNA binding of the CDC2 cell-cycle promoter, shown to be functionally dependent from both E2F and NF-Y. While results obtained indicate non-cooperative DNA association of NF-Y and E2F1, suggesting alternative mechanisms to elicit transcriptional synergy, we observed efficient assembly of stable ternary complexes. Together, our results and tools obtained in this work will be instrumental for future structural studies involving multi-protein complex assembly on DNA, and for defining the molecular bases of TFs interactions involved in architectural organisation of gene promoters.
MANTOVANI, ROBERTO
Transcription Factor; NF-Y; E2F1; DP1; SAXS; Molecular Dynamics
Settore BIO/10 - Biochimica
Settore BIO/18 - Genetica
STRUCTURAL DETERMINANTS OF NF-Y INTERACTION WITH PARTNER TRANSCRIPTION FACTORS ON DNA / D. Saad ; tutor: N. Gnesutta ; co-tutor: R. Mantovani. - : . Dipartimento di Bioscienze, 2021 Apr 22. ((33. ciclo, Anno Accademico 2020.
Doctoral Thesis
File in questo prodotto:
File Dimensione Formato  
phd_unimi_R11982.pdf

embargo fino al 16/04/2022

Descrizione: Tesi di dottorato
Tipologia: Tesi di dottorato completa
Dimensione 10.59 MB
Formato Adobe PDF
10.59 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/836391
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact